The objectives of the proposed work are the determination of aspects of the structure of rabbit muscle phosphofructokinase and the elucidation of structure-function relationships for this important regulatory enzyme. The proposed work will include a) isolation and characterization of the two major fragments of "half-molecules" that result from limited proteolysis of native phosphofructokinase by trypsin, b) isolation and characterization of the sets of peptides produced by the further chemical cleavage of each of the "half molecules" at cysteine and methionine residues, c) determination of the locations of modifier and substrate binding sites and indentification of the segments of the polypeptide chain that comprise these sites by affinity-labeling the sites and d) investigation of the molecular basis for the regulation of phosphofructokinase through kinetic and structural studies of the enzyme containing affinity labels covalently attached at regulatory sites.